Enzymes are proteins that catalyze chemical reactions in vivo, and they have very specific catalytic activities toward chemical reaction and substrate. Enzyme reactions using their specific catalytic activities are utilized in medical and life science research. Enzymes find use in various catalytic chemical processes as well. In these utilizations of enzyme reactions, it is required in many cases to separate enzymes from reaction solutions after the reactions are completed, for the purpose of purifying products or recycling the enzymes. However, in solution phase, it takes long time and is very cumbersome to separate and purify materials such as enzymes and reaction products from reaction samples. Therefore, it is very important in the utilization of enzyme reactions to develop a method of simplifying the enzyme separation process.
Since immobilized enzymes can provide very efficient methods to simplify the enzyme separation process, a variety of work has been performed extensively on the development of methods for immobilizing enzymes. The “immobilized enzyme” as used herein means an enzyme that is physically or chemically bound to a substrate material while retaining substantial catalytic activity. Advantages of using such immobilized enzymes are as follows.
For instance, the purification or separation process of reaction samples can be simplified since the immobilized enzyme can be easily separated and recycled from reaction samples by simply removing the immobilized enzyme after an enzyme reaction. Also, the cost can be reduced by reusing the recycled immobilized enzyme. In addition, because multiple processes where enzyme reactions are included can be simplified, the efficiency of the overall reaction processes can be increased. Further, the efficiency of utilizing enzyme can be increased due to additional effects such as an enhancement in physical stability of enzyme after immobilization or a change in the conditions of enzyme reaction.
Examples of the enzyme immobilization methods include the carrier-binding method where enzymes bind to a carrier that is typically insoluble to water, the cross-linking method where enzymes are connected one another using a reagent having multiple reaction groups, and the entrapping method where enzymes are surrounded by semi-permeable gel or macromolecular membrane. The immobilized DNA polymerase in the present invention is prepared by a kind of the carrier-binding method where enzymes are immobilized on a substrate material with covalent bonding. The characteristics of the carrier-binding method are thus described in detail below.
In the carrier-binding method, the kind of a carrier and the method of binding are selected based on physical and chemical interactions between the enzyme and the carrier. This is because the amount and the characteristics of the enzyme after immobilization on the carrier depend greatly on the characteristics of the carrier and the method of binding. This method is thus classified according to the method of binding between the enzyme and the carrier, and examples of the carrier-binding method include the physical adsorption method via hydrogen bonding and Van der Waals interaction, the ionic bonding method where an enzyme binds to a polysaccharide having ion exchange groups or a synthetic macromolecule via ionic bonding, and the covalent bonding method where covalent bonding is formed between an enzyme and a carrier.
The physical adsorption method or the ionic bonding method has an advantage that the possibility of damaging the enzyme activity by immobilization bonding is low, since the enzyme binding process to a carrier is relatively simple and the influence on the active site structure of the enzyme is relatively low due to weak binding. However, there is a disadvantage that the enzyme can be lost even with the small change in temperature and pH, due to the weak binding between the enzyme and the carrier, and the low specificity that causes nonspecific binding of undesired proteins.
The disadvantage of the above two method can be solved in the covalent bonding method, since in the covalent bonding method, enzymes are immobilized by forming a strong bond between the enzyme and the carrier. Therefore, works on a variety of the covalent bonding methods have been performed using various kinds of carriers.
Since the enzyme has various reaction groups that can form covalent bond with a carrier, for example, amine, carboxyl, hydroxyl, thiol, imidazole, etc., immobilization reactions toward such reaction groups that can be performed in aqueous solution have been developed such as amide bond formation reaction, alkylation or arylation, disulfide bond formation, diazotization, etc. In addition, there has been reported an immobilization method where an immobilization reaction group on a substrate material reacts directly with a reaction group of an enzyme, or an immobilization method where a substrate material and an enzyme are connected using a linker having reaction groups at both ends. However, the covalent bonding method has a difficulty of preserving the enzyme activity after immobilization, since a strong bond formed between the enzyme and the carrier gives rise to the structural change in the enzyme which in turn increases the possibility of damaging the enzyme activity.
The most important point in immobilizing an enzyme on a substrate material with covalent bonding is that the damage of the enzyme activity due to a structural change in the enzyme active site by immobilization bonding must be prevented. Therefore, it is essential that the immobilization reaction must not occur at or near the active site. And also, even if the immobilization reaction occurs at a site distant from the active site of the enzyme, the allosteric effect that eventually reduces the enzyme activity by influencing the total structure of the enzyme must not occur. Therefore, in order to immobilize the enzyme with its activity preserved, it must be possible to select an immobilization site in the enzyme toward which immobilization bonding occurs, i.e., to select a reaction group of the enzyme located at a particular site such that the activity is not damaged after the immobilization bonding. In other words, an oriented immobilization should be possible wherein the enzyme can be immobilized in an oriented manner so that a reaction group of the enzyme located at the particular site forms an immobilization bonding. However, an enzyme is a macromolecule that has a great number of amino acids connected by amide bonding. Due to such characteristic of the enzyme, reaction groups available for immobilization reaction, for example, amine, carboxyl, hydroxyls, etc. are numerous and they are distributed throughout the enzyme. Therefore, it is impossible with the prior technologies developed to date to direct an oriented immobilization by selecting a reaction group of an enzyme located at a particular site. In other words, in the enzyme immobilization using the prior art immobilization methods, random immobilization reactions occur toward a plurality of reaction groups in the enzyme. Therefore, an immobilization bonding can be formed at an undesirable site or a plurality of immobilization bonding can be formed, thereby severely damaging the enzyme activity.
In addition to the damage in the enzyme activity due to the nonspecific nature of the immobilization reaction in the prior art immobilization methods, the formation of covalent bonding by itself can possibly reduce the enzyme activity largely because it requires rearrangement of electrons. That is, even if it is possible to select a reaction group of the enzyme located at a particular site so as to direct an oriented immobilization toward the reaction group, it is impossible to confirm presence of the covalently immobilized enzyme with its activity preserved without damage unless an oriented immobilization is actually performed.
There have been efforts to immobilize certain biomolecules other than DNA polymerase. For example, U.S. Pat. No. 4,180,383 discloses a method of making an immobilized immunoadsorbent in which an antigen is used as a masking agent to protect (mask) an antibody of interest before reaction with a polymer support. U.S. Pat. No. 6,194,552 and Subramanian A. and W. Velander (1996) J. of Mol. Recognition. 9: 528 also disclose a similar method for preparing an immobilized immunoadsorbent using an antigen as a masking agent. U.S. Pat. No. 6,172,202 (see also PCT/EP93/03429 (WO 94/13322) discloses a method for preparing a conjugate of a protein (or a glycoprotein) with a water soluble protein using an antibody or an antiidiotypic antibody as a masking agent.
However, there is general understanding that such methods work largely because antibodies are robust molecules. That is, even unmasked antibodies are thought to be less sensitive to conformational changes induced by immobilization reactions occurring outside the antigen binding pocket. In marked contrast, DNA polymerase is thought to be especially sensitive to reactions involving amino acid residues within and outside the catalytic site. In particular, the prolonged antigen binding conditions required by the prior masking procedures are believed to be particularly unsuitable for manipulating sensitive DNA polymerases.
More generally, there is increasing recognition that prior methods of protecting antibodies and certain proteins before reaction with a substrate material will not provide good results with DNA polymerases. There is doubt that current approaches that focus on protecting active sites will be insufficient to preserve the biological activity of a DNA polymerase following immobilization. In practice, it has been difficult or in some cases impossible to preserve the biological activity of DNA polymerase after an immobilization procedure.
It would be desirable to have a method of immobilizing a DNA polymerase to a substrate material. It would be further desirable to have methods that protect not only the catalytic site of the DNA polymerase but also optimize interaction of the enzyme with the substrate material.